CN105661008A - Method for producing protein feed from apple waste and cottonseed meal through mixed fermentation - Google Patents
Method for producing protein feed from apple waste and cottonseed meal through mixed fermentation Download PDFInfo
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- CN105661008A CN105661008A CN201610052430.7A CN201610052430A CN105661008A CN 105661008 A CN105661008 A CN 105661008A CN 201610052430 A CN201610052430 A CN 201610052430A CN 105661008 A CN105661008 A CN 105661008A
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Abstract
The invention discloses a method for producing protein feed from apple waste and cottonseed meal through mixed fermentation. The method for producing the protein feed from the apple waste and the cottonseed meal through mixed fermentation comprises steps as follows: 1, preparation of an enterococcus faecalis R-F bacterial liquid: the preparation comprises steps of bacterium activation, shake-flask bacterium culture, bacterium tank culture and fermentation tank culture; 2, specific fermentation: the apple waste, the cottonseed meal and bran are mixed and blended with protease, cellulose, pectinase, light calcium carbonate, molasses and the enterococcus faecalis R-F bacterial liquid, water is added to regulate the water content of the materials in 30%-45%, and a mixture is placed in a check-valve fermentation bag and subjected to fermentation for 5-15 days in a fermentation chamber at the temperature of 15-30 DEG C. After fermentation with the method, the number of live bacteria in the feed is up to 2 billion/g, the content of the crude protein is higher than or equal to 15%, the content of small peptides is higher than or equal to 10%, the content of pectin is lower than or equal to 5%, the content of tannin is higher than or equal to 0.02%, and the content of free gossypol is lower than or equal to 25 mg/kg.
Description
Technical field
The present invention relates to biological feedstuff fermentation arts, be specifically related to a kind of enterococcus faecalis bacterium solution preparation and fermentation apple pomace thereof and the method for cottonseed meal production albumen feedstuff.
Background technology
China is fruit big producing country of the world, occupies first place in the world in total output position. 1998 year outputs reach 5452.9 ten thousand tons, wherein Fructus Mali pumilae 1948.1 ten thousand tons, occupy No. 1 in the world, and working ability only has about 5%. The main producing region for pomace is economized in China Shandong, Hebei, Shanxi, Shaanxi etc. 2006, China's cultivated area of the apple 1,900,000 hectares, yield 26,000,000 tons, produce more than the 35% of world wide production.
Developing rapidly of fruit industry, converted products is of a great variety, and working ability improves constantly, and meanwhile, the output of pomace also increases increasingly. According to statistics, in the end of the year 2007, Shanxi Province's Apples ability is about 1,860,000 tons, and large-scale concentrated Succus Mali pumilae processing enterprise reaches 12, annual working ability 22.6 ten thousand tons. But the processing and utilization of pomace, does not receive attention, not only to environment, but also cause the waste of resource. Pomace is high-moisture material, containing part soluble sugar, vitamin, mineral and cellulose, has an abundant nutrient substance, and major part Fruit quality factory using marc as offal treatment, cause the significant wastage of resource, also have a strong impact on environment. Containing the antinutritional factor such as tannin, pectin in pomace, limit the use of pomace.
Shi Chanmian big country of China, cotton cake dregs yield is big, annual output about 3,000,000 tons, is rich in protein source. The cottonseed meal crude protein that shelling degree is higher can reach more than 50%, and the Semen Gossypii of common Cotton Gossypii contains the gossypol with toxicity and derivant thereof, is commonly called as cotton toxin, nonruminant is harmful to, and tradition poison-removing method efficiency is low, cost is high, has had a strong impact on cottonseed meal application in monogastric animal feed.
Summary of the invention
It is an object of the invention to the fermentation through microorganism, the antinutritional factor in degraded pomace and cottonseed meal, improve protein and little peptide content in feedstuff simultaneously, produce a kind of albumen feedstuff.
The present invention adopts the following technical scheme that realization:
A kind of method that pomace and cottonseed meal mixed fermentation produce albumen feedstuff, comprises the following steps:
, preparation in enterococcus faecalis R-F bacterium solution
(1), the activation of strain: the enterococcus faecalis R-F that inclined-plane preserves is activated 3 times on activation medium, single bacterium colony that on picking MRS flat board, transparent circle is bigger, access deep layer MRS liquid tube, after 30-35 DEG C of cultivation 12-16h, access MRS fluid medium with 1% inoculum concentration, cultivate 18-24h as seed liquor;
(2), shake-flask seed is cultivated:
One-level shake-flask seed is cultivated: being accessed in one-level Shake flask medium according to the inoculum concentration of 5-10% by the enterococcus faecalis R-F activated, 30-35 DEG C of quiescent culture 12-16h, as one-level shake-flask seed liquid;
Second-level shake flask seed culture: one-level shake-flask seed liquid is linked in second-level shake flask culture medium according to the inoculum concentration of 5-10%, quiescent culture 16-20h in 30-35 DEG C of incubator, as second-level shake flask seed liquor;
(3), seed tank culture:
First class seed pot is cultivated: receive in first class seed pot culture medium according to 2-5% inoculum concentration by second-level shake flask seed liquor, cultivation temperature 30-35 DEG C, tank pressure 0.03-0.05MPa, stirs cultivation 12-18h under 100-200 rev/min of condition, treats that total bacteria count reaches 1-3 × 109During more than CFU/ml, proceed in secondary seed tank culture medium and cultivate;
Secondary seed tank is cultivated: receive in secondary seed tank culture medium according to 5-8% inoculum concentration by first class seed pot seed liquor, cultivation temperature 30-35 DEG C, tank pressure 0.03-0.05MPa, stirs cultivation 14-20h under 100-200 rev/min of condition, treats that total bacteria count reaches 4-7 × 109During more than CFU/ml, proceed to fermentation tank and cultivate;
(4), fermentor cultivation:
According to the inoculum concentration of 5-10%, secondary seed tank seed liquor is proceeded to the fermentation basal medium of fermentation tank, at temperature 30-35 DEG C, stirs 100-120 rev/min, ferment when tank pressure 0.03-0.05MPa, in sweat, regulating pH at 6.0-6.5, incubation time is 22-24 hour;
Wherein, in enterococcus faecalis R-F bacterium solution preparation step (1), the composition of activation medium includes: peptone 5-15g/L, Carnis Bovis seu Bubali cream 5-15g/L, yeast leaching powder 3-10g/L, glucose 10-25g/L, dibasic ammonium citrate 1-4g/L, sodium acetate 2-7g/L, dipotassium hydrogen phosphate 1-5g/L, Magnesium sulfate heptahydrate 0.25-0.65g/L, manganese sulfate monohydrate 0.1-0.3g/L, pH6.0-6.5.
In enterococcus faecalis R-F bacterium solution preparation step (2), the composition of one-level Shake flask medium includes: peptone 5-15g/L, Carnis Bovis seu Bubali cream 5-15g/L, yeast leaching powder 3-10g/L, glucose 10-25g/L, dibasic ammonium citrate 1-4g/L, sodium acetate 2-7g/L, dipotassium hydrogen phosphate 1-5g/L, Magnesium sulfate heptahydrate 0.25-0.65g/L, manganese sulfate monohydrate 0.1-0.3g/L, pH6.0-6.5. The composition of second-level shake flask culture medium includes: soybean cake powder 15-25g/L, peptone 5-15g/L, yeast leaching powder 5-15g/L, glucose 15-25g/L, calcium carbonate 0.5-1.5g/L, dibasic ammonium citrate 1-4g/L, sodium acetate 2-7g/L, dipotassium hydrogen phosphate 1-5g/L, Magnesium sulfate heptahydrate 0.25-0.65g/L, manganese sulfate monohydrate 0.1-0.3g/L, pH6.0-6.5.
In enterococcus faecalis R-F bacterium solution preparation step (3), the composition of first class seed pot culture medium includes: soybean cake powder 25-35g/L, peptone 5-15g/L, yeast leaching powder 5-15g/L, glucose 15-25g/L, calcium carbonate 0.5-1.5g/L, dibasic ammonium citrate 1-4g/L, sodium acetate 2-7g/L, dipotassium hydrogen phosphate 1-5g/L, Magnesium sulfate heptahydrate 0.25-0.65g/L, manganese sulfate monohydrate 0.1-0.3g/L, pH6.0-6.5.The composition of secondary seed tank culture medium includes: soybean cake powder 25-35g/L, peptone 5-15g/L, yeast leaching powder 5-15g/L, glucose 15-25g/L, calcium carbonate 0.5-1.5g/L, dibasic ammonium citrate 1-4g/L, sodium acetate 2-7g/L, dipotassium hydrogen phosphate 1-5g/L, Magnesium sulfate heptahydrate 0.25-0.65g/L, manganese sulfate monohydrate 0.1-0.3g/L, pH6.0-6.5.
In enterococcus faecalis R-F bacterium solution preparation step (4), fermentation basic media components includes: soybean cake powder 25-35g/L, peptone 5-15g/L, yeast leaching powder 5-15g/L, glucose 15-25g/L, calcium carbonate 0.5-1.5g/L, dibasic ammonium citrate 1-4g/L, sodium acetate 2-7g/L, dipotassium hydrogen phosphate 1-5g/L, Magnesium sulfate heptahydrate 0.25-0.65g/L, manganese sulfate monohydrate 0.1-0.3g/L, pH6.0-6.5. Further, fermentation to fermentation basal medium residual sugar content below 0.8% time, mend sugar with fermentation medium supplementing culture medium, mend after sugar residual sugar in culture fluid and control at 1-2%. Fermentation medium supplementing culture medium is glucose solution: in described glucose solution, glucose content is the 2-4% of fermentation liquid total amount
, concrete fermentation step:
By pomace, cottonseed meal, wheat bran mixing, being equipped with protease, cellulase, pectase, precipitated calcium carbonate, molasses and enterococcus faecalis R-F bacterium solution, the adjustment material moisture that adds water, at 30-45%, is placed in round, in 15-30 DEG C of fermenting cellar, fermenting 5-15 days, in feedstuff, viable count reaches 2,000,000,000/more than g, crude protein >=15%, little peptide >=10%, pectin≤5%, tannin≤0.02%, free gossypol content≤25mg/kg. After fermentable, the antinutritional factor degradation rate such as tannin and pectin respectively reaches 99.0% and 66.1%, and crude protein and little peptide respectively reach 13-15% and 8-10%, and free gossypol content reduces by more than 80%. By the palatability improving pomace and cottonseed meal of fermenting, antinutritional factor significantly reduces, and improves the feeding value of pomace and cottonseed meal to greatest extent.
Preferably, stepIn, constitute mixture after the ratio of pomace 20-80 part, cottonseed meal 1-40 part, wheat bran 1-30 part, precipitated calcium carbonate 0.5-5 part being mixed, wherein, total number of pomace, cottonseed meal, wheat bran and precipitated calcium carbonate is 100; Again with mixture weight for counting, add cellulase 0.05-0.07%, proteinase-10 .03-0.05%, pectase 0.02-0.07%, molasses 0.02-0.3%, enterococcus faecalis R-F bacterium solution 8-20%.
Wherein, described organized enzyme is:
A, protease, enzyme activity >=105U/g, addition is 300 ~ 500g/t.
B, cellulase, enzyme activity >=105U/g, addition 500 ~ 700g/t.
C, pectase, enzyme activity >=30000U/g, addition 200 ~ 700g/t.
The present invention adopts check valve fermentation bag to ferment, effectively stop the pollution of miscellaneous bacteria in sweat, in Enterococcus faecalis fermentation process, utilize pomace and protein rich cottonseed meal that carbohydrate containing is abundant, the antinutritional factor such as the gossypol in the tannin, pectin and the cottonseed meal that reduce in pomace by fermenting, enterococcus faecalis utilizes pomace, cottonseed meal, wheat bran, molasses to breed simultaneously, and the content of probiotics increases. Meanwhile, after the fermentation of marc and cottonseed meal, lactic acid content increases, and has quality softness, holding time length, with low cost, it is possible to be effectively improved its utilization rate.
Adopt the fermentation of apple pulp feedstuff that the present invention produces, animal and fowl fodder conversion ratio can be made to improve 8-15%, crude protein improves 3 ~ 5%, little peptide improves 50 ~ 80%, enterococcus faecalis contained in fermented feed, can effectively regulating animal and bird intestines microecological balance, prevention animal diarrhea, raising daily gain, in poultry complete feedstuff, addition is up to 5-10%.
Detailed description of the invention
Below specific embodiments of the invention are described in detail.
Embodiment 1
A kind of method that pomace and cottonseed meal mixed fermentation produce albumen feedstuff, comprises the following steps:
, enterococcus faecalis R-F bacterium solution preparation
(1), the activation of strain: for improving the vigor of strain, before connecing shaking flask, it is necessary to strain is activated. After the enterococcus faecalis R-F normal saline dilution that inclined-plane is preserved, coat on MRS solid (activation medium) flat board, cultivate 24h for 35 DEG C, single bacterium colony that picking transparent circle is bigger, carry out plate isolation line, cultivate 24h for 35 DEG C, the bigger single bacterium colony of picking transparent circle is on MRS solid plate, cultivate 24h for 35 DEG C, finally, picking transparent circle relatively macrocolony accesses deep layer MRS liquid tube, 35 DEG C of static gas wave refrigerator 12h, access in MRS fluid medium according to 1% inoculum concentration, cultivate 18h as seed liquor. Wherein, enterococcus faecalis R-F activation culture based formulas is: peptone 10g/L, Carnis Bovis seu Bubali cream 10g/L, yeast leaching powder 5g/L, glucose 20g/L, dibasic ammonium citrate 2g/L, sodium acetate 5g/L, dipotassium hydrogen phosphate 2g/L, Magnesium sulfate heptahydrate 0.25/L, manganese sulfate monohydrate 0.2g/L, pH6.0-6.5.
(2), shake-flask seed is cultivated:
One-level shake-flask seed is cultivated: according to the inoculum concentration of 5%, being accessed in one-level Shake flask medium by the enterococcus faecalis R-F activated, 250ml triangular flask liquid amount 100ml, at 35 DEG C of static gas wave refrigerator 12h. One-level shake-flask culture based formulas is: peptone 12g/L, Carnis Bovis seu Bubali cream 8g/L, yeast leaching powder 5g/L, glucose 20g/L, dibasic ammonium citrate 2g/L, sodium acetate 5g/L, dipotassium hydrogen phosphate 2g/L, Magnesium sulfate heptahydrate 0.58g/L, manganese sulfate monohydrate 0.25g/L, pH6.0-6.5.
Second-level shake flask seed culture: according to the inoculum concentration of 8%, is linked in second-level shake flask culture medium by one-level shake-flask seed liquid, 500ml triangular flask liquid amount 250ml, quiescent culture 16h in 33 DEG C of incubators. Second-level shake flask seed culture based formulas is: soybean cake powder 15g/L, peptone 10g/L, yeast leaching powder 10g/L, glucose 15g/L, calcium carbonate 0.5g/L, dibasic ammonium citrate 2g/L, sodium acetate 5g/L, dipotassium hydrogen phosphate 2g/L, Magnesium sulfate heptahydrate 0.58g/L, manganese sulfate monohydrate 0.25/L, pH6.0-6.5, shaking flask terminate after as one grade fermemtation tank seed liquor.
(3), seed tank culture:
First class seed pot seed culture: the seed liquor of second-level shake flask is accessed in first class seed pot by the inoculum concentration according to 3%, and the canned liquid coefficient of first order seed is 50%, cultivation temperature 35 DEG C, rotating speed 120 revs/min, tank pressure 0.03Mpa, cultivates 12h with this understanding, treats that total bacteria count reaches 1.5 × 109During more than CFU/ml, proceed to secondary seed tank and cultivate. Wherein, first class seed pot culture medium prescription is: soybean cake powder 25g/L, peptone 10g/L, yeast leaching powder 10g/L, glucose 15g/L, calcium carbonate 1.0g/L, dibasic ammonium citrate 2g/L, sodium acetate 5g/L, dipotassium hydrogen phosphate 2g/L, Magnesium sulfate heptahydrate 0.58g/L, manganese sulfate monohydrate 0.25/L, pH6.0-6.5.
Secondary seed tank seed culture: the strain in first class seed pot is accessed in secondary seed tank by the inoculum concentration according to 5%, and the canned liquid coefficient of secondary seed is 55%, and cultivation temperature is 35 DEG C, rotating speed 130 revs/min, tank pressure 0.05Mpa, cultivates 16h with this understanding, treats that total bacteria count reaches 5.0 × 109During more than CFU/ml, proceed to fermentation tank and cultivate. Secondary seed tank seed culture based formulas is: soybean cake powder 25g/L, peptone 8g/L, yeast leaching powder 5g/L, glucose 20g/L, calcium carbonate 0.5g/L, ammonium citrate 3g/L, sodium acetate 4g/L, dipotassium hydrogen phosphate 3g/L, Magnesium sulfate heptahydrate 0.4g/L, manganese sulfate monohydrate 0.25g/L, pH6.0-6.5.
(4), fermentor cultivation:
Fermentation tank High Density Cultivation: secondary seed tank bacterium solution is accessed in fermentation tank according to 10% inoculum concentration, the canned liquid coefficient that ferments is 60%, cultivation temperature is 35 DEG C, rotating speed 120 revs/min, tank pressure 0.05Mpa, cultivate about 3 hours with this understanding, when fermentation liquid residual sugar drops to below 0.8%, mend sugar once per hour, after mending sugar, culture fluid residual sugar controls below 2%, carrying out along with fermentation, enterococcus faecalis produces acid, fermentation liquid pH declines, when fermentation liquid pH is lower than 6.0, the NaOH of auto-feeding 2mol/L, supplement alkali liquor and regulate pH to 6.0-6.5, incubation time is 22-24 hour. fermentation basal medium formulation is: soybean cake powder 30g/L, peptone 8g/L, yeast leaching powder 5g/L, glucose 20g/L, calcium carbonate 1.0g/L, ammonium citrate 3g/L, sodium acetate 4g/L, dipotassium hydrogen phosphate 3g/L, Magnesium sulfate heptahydrate 0.4g/L, manganese sulfate monohydrate 0.25g/L. fermentation medium feed supplement formula is: Glucose Liquid cumulative volume is the 4% of fermentation tank, and concentration of glucose is 50%, with fermentation tank culture medium according to same condition sterilizing before using.
Enterococcus faecalis R-F cultivates to fermentation tank through overactivation, shaking flask, seed tank, coordinates feed supplement and regulation and control pH in sweat, and final enterococcus faecalis R-F cell concentration is up to 1.0 × 1010More than CFU/ml.
, fermentation of apple pulp processing step:
(1), pulverize: with pulverizer, dry fruit slag, cottonseed meal, wheatfeed are broken into the granule of 0.8mm.
(2) by dry pomace 60 parts and cottonseed meal 30 parts, 9 parts of wheat bran, the ratio mixing that precipitated calcium carbonate is 1 part, again by cellulase, protease, pectase, molasses, enterococcus faecalis R-F bacterium solution according to 0.05%, 0.05%, 0.05%, 0.05%, 10%(account for dry matter weight ratio) be added to the water, regulating amount of water makes material-water ratio be 1:0.5, finally being sprayed on material by water, uniform stirring, final material moisture is about 40%. Wherein, cellulase, protease, pectase enzyme live respectively 100,000 U/g, 100,000 U/g, 30,000 U/g.
(3), the material being stirred is joined with in check valve fermentation bag, then fermenting 5-15 days in the fermenting cellar of 15 DEG C, 72h samples once, detection viable count, crude protein, little peptide and gossypol content, sample one-time detection These parameters every 48h later, treat that viable count reaches 2,000,000,000/g, crude protein >=13%, little peptide >=8%, pectin≤5%, tannin≤0.02%, during gossypol content≤25mg/kg, product is qualified.
Embodiment 2
A kind of method that pomace and cottonseed meal mixed fermentation produce albumen feedstuff, comprises the following steps:
, enterococcus faecalis R-F bacterium solution preparation
(1), the activation of strain: for improving the vigor of strain, before connecing shaking flask, it is necessary to strain is activated. After the enterococcus faecalis R-F normal saline dilution that inclined-plane is preserved, coat on MRS solid plate, cultivate 24h, single bacterium colony that picking transparent circle is bigger for 33 DEG C, carry out plate isolation line, cultivate 24h for 30 DEG C, the bigger single bacterium colony of picking transparent circle is on MRS solid plate, cultivate 24h for 33 DEG C, last picking transparent circle relatively macrocolony accesses deep layer MRS liquid tube, 33 DEG C of static gas wave refrigerator 14h, access in MRS fluid medium according to 1% inoculum concentration, cultivate 20h as seed liquor. Wherein, enterococcus faecalis R-F activation culture based formulas is: peptone 5g/L, Carnis Bovis seu Bubali cream 15g/L, yeast leaching powder 10g/L, glucose 10g/L, dibasic ammonium citrate 1g/L, sodium acetate 7g/L, dipotassium hydrogen phosphate 1g/L, Magnesium sulfate heptahydrate 0.45/L, manganese sulfate monohydrate 0.3g/L, pH6.0-6.5.
(2), shake-flask seed is cultivated:
One-level shake-flask seed is cultivated: according to the inoculum concentration of 8%, the slant strains after activation being received one-level Shake flask medium, 250ml triangular flask liquid amount 100ml, at 33 DEG C of static gas wave refrigerator 16h. One-level shake-flask culture based formulas is: peptone 10g/L, Carnis Bovis seu Bubali cream 10g/L, yeast leaching powder 10g/L, glucose 15g/L, dibasic ammonium citrate 3g/L, sodium acetate 4g/L, dipotassium hydrogen phosphate 3g/L, Magnesium sulfate heptahydrate 0.40g/L, manganese sulfate monohydrate 0.15g/L, pH6.0-6.5.
Second-level shake flask seed culture: according to the inoculum concentration of 5%, is linked in second-level shake flask culture medium by one-level shake-flask seed, 500ml triangular flask liquid amount 250ml, quiescent culture 20h in 35 DEG C of incubators. Second-level shake flask seed culture based formulas is: soybean cake powder 20g/L, peptone 8g/L, yeast leaching powder 8g/L, glucose 20g/L, calcium carbonate 1.0g/L, dibasic ammonium citrate 3g/L, sodium acetate 4g/L, dipotassium hydrogen phosphate 3g/L, Magnesium sulfate heptahydrate 0.40g/L, manganese sulfate monohydrate 0.15/L, pH6.0-6.5, shaking flask terminate after as one grade fermemtation tank seed liquor.
(3), seed tank culture:
First class seed pot seed culture: the seed liquor of second-level shake flask is accessed in first class seed pot by the inoculum concentration according to 5%, and the canned liquid coefficient of first order seed is 50%, cultivation temperature 33 DEG C, rotating speed 100 revs/min, tank pressure 0.05Mpa, cultivates 15h with this understanding, treats that total bacteria count reaches 1.5 × 109During more than CFU/ml, proceed to secondary seed tank and cultivate. Wherein, first class seed pot culture medium prescription is: soybean cake powder 30g/L, peptone 8g/L, yeast leaching powder 8g/L, glucose 20g/L, calcium carbonate 0.5g/L, dibasic ammonium citrate 3g/L, sodium acetate 4g/L, dipotassium hydrogen phosphate 3g/L, Magnesium sulfate heptahydrate 0.40g/L, manganese sulfate monohydrate 0.1g/L, pH6.0-6.5.
Secondary seed tank seed culture: the strain in first class seed pot is accessed in secondary seed tank by the inoculum concentration according to 6%, and the canned liquid coefficient of secondary seed is 55%, and cultivation temperature is 33 DEG C, rotating speed 200 revs/min, tank pressure 0.04Mpa, cultivates 20h with this understanding, treats that total bacteria count reaches 5.0 × 109During more than CFU/ml, proceed to fermentation tank and cultivate. Secondary seed tank seed culture based formulas is: soybean cake powder 35g/L, peptone 6g/L, yeast leaching powder 10g/L, glucose 15g/L, calcium carbonate 1.5g/L, ammonium citrate 1g/L, sodium acetate 2g/L, dipotassium hydrogen phosphate 5g/L, Magnesium sulfate heptahydrate 0.25g/L, manganese sulfate monohydrate 0.1g/L, pH6.0-6.5.
(4), fermentor cultivation:
Fermentation tank High Density Cultivation: secondary seed tank bacterium solution is accessed in fermentation tank according to 5% inoculum concentration, the canned liquid coefficient that ferments is 60%, cultivation temperature is 33 DEG C, rotating speed 100 revs/min, tank pressure 0.03Mpa, cultivates about 3 hours with this understanding, when fermentation liquid residual sugar drops to below 0.8%, mending sugar once per hour, after mending sugar, culture fluid residual sugar controls below 2%. Along with the carrying out of fermentation, enterococcus faecalis produces acid, and fermentation liquid pH declines, when fermentation liquid pH is lower than 6.0, and the NaOH of auto-feeding 2mol/L, supplement alkali liquor and regulate pH to 6.0-6.5, incubation time is 22-24 hour. Fermentor cultivation based formulas is: soybean cake powder 25g/L, peptone 6g/L, yeast leaching powder 10g/L, glucose 25g/L, calcium carbonate 0.5g/L, ammonium citrate 4g/L, sodium acetate 2g/L, dipotassium hydrogen phosphate 1g/L, Magnesium sulfate heptahydrate 0.25g/L, manganese sulfate monohydrate 0.1g/L.Fermentation medium feed supplement formula is: Glucose Liquid cumulative volume is the 4% of fermentation tank, and concentration of glucose is 50%, with fermentation tank culture medium according to same condition sterilizing before using.
Enterococcus faecalis R-F cultivates to fermentation tank through overactivation, shaking flask, seed tank, coordinates feed supplement and regulation and control pH in sweat, and final enterococcus faecalis R-F cell concentration is up to 1.0 × 1010More than CFU/ml.
, fermentation of apple pulp processing step:
(1), pulverize: with pulverizer, dry fruit slag, cottonseed meal, wheatfeed are broken into the granule of 1.0mm.
(2), by dry pomace 25 parts and cottonseed meal 40 parts, 30 parts of wheat bran, the ratio mixing that precipitated calcium carbonate is 5 parts, again by cellulase, protease, pectase, molasses, enterococcus faecalis R-F bacterium solution according to 0.06%, 0.03%, 0.02%, 0.1%, 12%(account for dry matter weight ratio) be added to the water, regulating amount of water makes material-water ratio be 1:0.5, finally being sprayed on material by water, uniform stirring, final material moisture is about 40%. Wherein, cellulase, protease, pectase enzyme live respectively 100,000 U/g, 100,000 U/g, 30,000 U/g.
(3), the material being stirred is joined with in check valve fermentation bag, then ferment 5-15 days in the fermenting cellar of 30 DEG C, 72h samples once, detection viable count, crude protein, little peptide and gossypol content, samples one-time detection These parameters every 48h later, treat that viable count reaches 2,000,000,000/g, crude protein >=15%, little peptide >=10%, pectin≤5%, tannin≤0.02%, during free gossypol content≤25mg/kg, product is qualified.
Embodiment 3
A kind of method that pomace and cottonseed meal mixed fermentation produce albumen feedstuff, comprises the following steps:
, enterococcus faecalis R-F bacterium solution preparation
(1), the activation of strain: for improving the vigor of strain, before connecing shaking flask, it is necessary to strain is activated. After the enterococcus faecalis R-F normal saline dilution that inclined-plane is preserved, coat on MRS solid plate, cultivate 24h, single bacterium colony that picking transparent circle is bigger for 33 DEG C, carry out plate isolation line, cultivate 24h for 30 DEG C, the bigger single bacterium colony of picking transparent circle is on MRS solid plate, cultivate 24h for 33 DEG C, last picking transparent circle relatively macrocolony accesses deep layer MRS liquid tube, 30 DEG C of static gas wave refrigerator 16h, access in MRS fluid medium according to 1% inoculum concentration, cultivate 24h as seed liquor. Wherein, enterococcus faecalis R-F activation culture based formulas is: peptone 15g/L, Carnis Bovis seu Bubali cream 5g/L, yeast leaching powder 3g/L, glucose 25g/L, dibasic ammonium citrate 4g/L, sodium acetate 2g/L, dipotassium hydrogen phosphate 5g/L, Magnesium sulfate heptahydrate 0.65/L, manganese sulfate monohydrate 0.1g/L, pH6.0-6.5.
(2), shake-flask seed is cultivated:
One-level shake-flask seed is cultivated: according to the inoculum concentration of 10%, the slant strains after activation being received one-level Shake flask medium, 250ml triangular flask liquid amount 100ml, at 30 DEG C of static gas wave refrigerator 14h. One-level shake-flask culture based formulas is: peptone 15g/L, Carnis Bovis seu Bubali cream 15g/L, yeast leaching powder 3g/L, glucose 25g/L, dibasic ammonium citrate 4g/L, sodium acetate 2g/L, dipotassium hydrogen phosphate 5g/L, Magnesium sulfate heptahydrate 0.25g/L, manganese sulfate monohydrate 0.3g/L, pH6.0-6.5.
Second-level shake flask seed culture: according to the inoculum concentration of 10%, is linked in second-level shake flask culture medium by one-level shake-flask seed, 500ml triangular flask liquid amount 250ml, quiescent culture 18h in 30 DEG C of incubators. Second-level shake flask seed culture based formulas is: soybean cake powder 25g/L, peptone 5g/L, yeast leaching powder 15g/L, glucose 25g/L, calcium carbonate 1.5g/L, dibasic ammonium citrate 4g/L, sodium acetate 7g/L, dipotassium hydrogen phosphate 1g/L, Magnesium sulfate heptahydrate 0.65g/L, manganese sulfate monohydrate 0.3/L, pH6.0-6.5, shaking flask terminate after as one grade fermemtation tank seed liquor.
(3), seed tank culture:
First class seed pot seed culture: the seed liquor of second-level shake flask is accessed in first class seed pot by the inoculum concentration according to 2%, and the canned liquid coefficient of first order seed is 50%, cultivation temperature 30 DEG C, rotating speed 200 revs/min, tank pressure 0.04Mpa, cultivates 18h with this understanding, treats that total bacteria count reaches 1.5 × 109During more than CFU/ml, proceed to secondary seed tank and cultivate. Wherein, first class seed pot culture medium prescription is: soybean cake powder 35g/L, peptone 15g/L, yeast leaching powder 15g/L, glucose 25g/L, calcium carbonate 1.5g/L, dibasic ammonium citrate 4g/L, sodium acetate 2g/L, dipotassium hydrogen phosphate 1g/L, Magnesium sulfate heptahydrate 0.25g/L, manganese sulfate monohydrate 0.3g/L, pH6.0-6.5.
Secondary seed tank seed culture: the strain in first class seed pot is accessed in secondary seed tank by the inoculum concentration according to 8%, and the canned liquid coefficient of secondary seed is 55%, and cultivation temperature is 30 DEG C, rotating speed 100 revs/min, tank pressure 0.03Mpa, cultivates 20h with this understanding, treats that total bacteria count reaches 5.0 × 109During more than CFU/ml, proceed to fermentation tank and cultivate. Secondary seed tank seed culture based formulas is: soybean cake powder 30g/L, peptone 15g/L, yeast leaching powder 15g/L, glucose 25g/L, calcium carbonate 1.0g/L, dibasic ammonium citrate 4g/L, sodium acetate 7g/L, dipotassium hydrogen phosphate 1g/L, Magnesium sulfate heptahydrate 0.65g/L, manganese sulfate monohydrate 0.3g/L, pH6.0-6.5.
(4), fermentor cultivation:
Fermentation tank High Density Cultivation: secondary seed tank bacterium solution is accessed in fermentation tank according to 8% inoculum concentration, the canned liquid coefficient that ferments is 60%, cultivation temperature is 30 DEG C, rotating speed 100 revs/min, tank pressure 0.04Mpa, cultivates about 3 hours with this understanding, when fermentation liquid residual sugar drops to below 0.8%, mending sugar once per hour, after mending sugar, culture fluid residual sugar controls below 2%. Along with the carrying out of fermentation, enterococcus faecalis produces acid, and fermentation liquid pH declines, when fermentation liquid pH is lower than 6.0, and the NaOH of auto-feeding 2mol/L, supplement alkali liquor and regulate pH to 6.5-7.0, incubation time is 22-24 hour. Fermentor cultivation based formulas is: soybean cake powder 35g/L, peptone 15g/L, yeast leaching powder 15g/L, glucose 15g/L, calcium carbonate 1.5g/L, dibasic ammonium citrate 1g/L, sodium acetate 7g/L, dipotassium hydrogen phosphate 5g/L, Magnesium sulfate heptahydrate 0.65g/L, manganese sulfate monohydrate 0.3g/L. Fermentation medium feed supplement formula is: Glucose Liquid cumulative volume is the 4% of fermentation tank, and concentration of glucose is 50%, with fermentation tank culture medium according to same condition sterilizing before using.
Enterococcus faecalis R-F cultivates to fermentation tank through overactivation, shaking flask, seed tank, coordinates feed supplement and regulation and control pH in sweat, and final enterococcus faecalis R-F cell concentration is up to 1.0 × 1010More than CFU/ml.
, fermentation of apple pulp processing step:
(1), pulverize: with pulverizer, dry fruit slag, cottonseed meal, wheatfeed are broken into the granule of 1.0mm.
(2), by dry pomace 80 parts and cottonseed meal 10 parts, 9.5 parts of wheat bran, the ratio mixing that precipitated calcium carbonate is 0.5 part, again by cellulase, protease, pectase, molasses, enterococcus faecalis R-F bacterium solution according to 0.07%, 0.04%, 0.07%, 0.3%, 20%(account for dry matter weight ratio) be added to the water, regulating amount of water makes material-water ratio be 1:0.5, finally being sprayed on material by water, uniform stirring, final material moisture is about 40%. Wherein, cellulase, protease, pectase enzyme live respectively 100,000 U/g, 100,000 U/g, 30,000 U/g.
(3), the material being stirred is joined with in check valve fermentation bag, then ferment 5-15 days in the fermenting cellar of 33 DEG C, 72h samples once, detection viable count, crude protein, little peptide and gossypol content, samples one-time detection These parameters every 48h later, treat that viable count reaches 2,000,000,000/g, crude protein >=15%, little peptide >=10%, pectin≤5%, tannin≤0.02%, during free gossypol content≤25mg/kg, product is qualified.
It should be noted last that; above example is only in order to illustrate technical scheme and unrestricted; although the present invention being described in detail with reference to embodiment; it will be understood by those within the art that; technical scheme is modified or equivalent replacement; without departure from the spirit and scope of technical scheme, it all should be contained in the claims of the present invention.
Claims (10)
1. the method that a pomace and cottonseed meal mixed fermentation produce albumen feedstuff, it is characterised in that: comprise the following steps:
, preparation in enterococcus faecalis R-F bacterium solution
(1), the activation of strain: the enterococcus faecalis R-F that inclined-plane preserves is activated 3 times on activation medium, single bacterium colony that on picking MRS flat board, transparent circle is bigger, access deep layer MRS liquid tube, after 30-35 DEG C of cultivation 12-16h, access MRS fluid medium with 1% inoculum concentration, cultivate 18-24h as seed liquor;
(2), shake-flask seed is cultivated:
One-level shake-flask seed is cultivated: being accessed in one-level Shake flask medium according to the inoculum concentration of 5-10% by the enterococcus faecalis R-F activated, 30-35 DEG C of quiescent culture 12-16h, as one-level shake-flask seed liquid;
Second-level shake flask seed culture: one-level shake-flask seed liquid is linked in second-level shake flask culture medium according to the inoculum concentration of 5-10%, quiescent culture 16-20h in 30-35 DEG C of incubator, as second-level shake flask seed liquor;
(3), seed tank culture:
First class seed pot is cultivated: receive in first class seed pot culture medium according to 2-5% inoculum concentration by second-level shake flask seed liquor, cultivation temperature 30-35 DEG C, tank pressure 0.03-0.05MPa, stirs cultivation 12-18h under 100-200 rev/min of condition, proceeds in secondary seed tank culture medium and cultivate;
Secondary seed tank is cultivated: receive in secondary seed tank culture medium according to 5-8% inoculum concentration by first class seed pot seed liquor, cultivation temperature 30-35 DEG C, tank pressure 0.03-0.05MPa, stirs cultivation 14-20h under 100-200 rev/min of condition, proceeds to fermentation tank and cultivate;
(4), fermentor cultivation:
According to the inoculum concentration of 5-10%, secondary seed tank seed liquor is proceeded to the fermentation basal medium of fermentation tank, at temperature 30-35 DEG C, stirs 100-120 rev/min, ferment when tank pressure 0.03-0.05MPa, in sweat, regulating pH at 6.0-6.5, incubation time is 22-24 hour;
, concrete fermentation step:
By pomace, cottonseed meal, wheat bran, precipitated calcium carbonate mixing, being equipped with protease, cellulase, pectase, molasses and enterococcus faecalis R-F bacterium solution, the adjustment material moisture that adds water, at 30-45%, is placed in round, in 15-30 DEG C of fermenting cellar, fermenting 5-15 days, in feedstuff, viable count reaches 2,000,000,000/more than g, crude protein >=15%, little peptide >=10%, pectin≤5%, tannin≤0.02%, free gossypol content≤25mg/kg.
2. the method that pomace according to claim 1 and cottonseed meal mixed fermentation produce albumen feedstuff, it is characterised in that: stepIn, constitute mixture after the ratio of pomace 20-80 part, cottonseed meal 1-40 part, wheat bran 1-30 part, precipitated calcium carbonate 0.5-5 part being mixed, wherein, total number of pomace, cottonseed meal, wheat bran and precipitated calcium carbonate is 100;Again with mixture weight for counting, add cellulase 0.05-0.07%, proteinase-10 .03-0.05%, pectase 0.02-0.07%, molasses 0.02-0.3%, enterococcus faecalis R-F bacterium solution 8-20%.
3. the method that pomace according to claim 2 and cottonseed meal mixed fermentation produce albumen feedstuff, it is characterised in that: protease, enzyme activity >=105U/g, addition is 300-500g/t; Cellulase, enzyme activity >=105U/g, addition 500-700g/t; Pectase, enzyme activity >=30000U/g, addition 200-700g/t.
4. the method that pomace according to claim 1 or 2 or 3 and cottonseed meal mixed fermentation produce albumen feedstuff, it is characterised in that: in step (1), described strain activation and culture based formulas is, peptone 5-15g/L, Carnis Bovis seu Bubali cream 5-15g/L, yeast leaching powder 3-10g/L, glucose 10-25g/L, dibasic ammonium citrate 1-4g/L, sodium acetate 2-7g/L, dipotassium hydrogen phosphate 1-5g/L, Magnesium sulfate heptahydrate 0.25-0.65g/L, manganese sulfate monohydrate 0.1-0.3g/L, pH6.0-6.5.
5. the method that pomace according to claim 1 or 2 or 3 and cottonseed meal mixed fermentation produce albumen feedstuff, it is characterized in that: in step (2), described one-level shake-flask culture based formulas is: peptone 5-15g/L, Carnis Bovis seu Bubali cream 5-15g/L, yeast leaching powder 3-10g/L, glucose 10-25g/L, dibasic ammonium citrate 1-4g/L, sodium acetate 2-7g/L, dipotassium hydrogen phosphate 1-5g/L, Magnesium sulfate heptahydrate 0.25-0.65g/L, manganese sulfate monohydrate 0.1-0.3g/L, pH6.0-6.5;
Described second-level shake flask culture medium prescription is: soybean cake powder 15-25g/L, peptone 5-15g/L, yeast leaching powder 5-15g/L, glucose 15-25g/L, calcium carbonate 0.5-1.5g/L, dibasic ammonium citrate 1-4g/L, sodium acetate 2-7g/L, dipotassium hydrogen phosphate 1-5g/L, Magnesium sulfate heptahydrate 0.25-0.65g/L, manganese sulfate monohydrate 0.1-0.3g/L, pH6.0-6.5.
6. the method that pomace according to claim 1 or 2 or 3 and cottonseed meal mixed fermentation produce albumen feedstuff, it is characterised in that: in step (3), the formula of described first class seed pot culture medium is: soybean cake powder 25-35g/L, peptone 5-15g/L, yeast leaching powder 5-15g/L, glucose 15-25g/L, calcium carbonate 0.5-1.5g/L, dibasic ammonium citrate 1-4g/L, sodium acetate 2-7g/L, dipotassium hydrogen phosphate 1-5g/L, Magnesium sulfate heptahydrate 0.25-0.65g/L, manganese sulfate monohydrate 0.1-0.3g/L, pH6.0-6.5;
The formula of described secondary seed tank culture medium is: soybean cake powder 25-35g/L, peptone 5-15g/L, yeast leaching powder 5-15g/L, glucose 15-25g/L, calcium carbonate 0.5-1.5g/L, dibasic ammonium citrate 1-4g/L, sodium acetate 2-7g/L, dipotassium hydrogen phosphate 1-5g/L, Magnesium sulfate heptahydrate 0.25-0.65g/L, manganese sulfate monohydrate 0.1-0.3g/L, pH6.0-6.5.
7. the method that pomace according to claim 1 or 2 or 3 and cottonseed meal mixed fermentation produce albumen feedstuff, it is characterised in that: in step (4), described fermentation basal medium formulation is: soybean cake powder 25-35g/L, peptone 5-15g/L, yeast leaching powder 5-15g/L, glucose 15-25g/L, calcium carbonate 0.5-1.5g/L, dibasic ammonium citrate 1-4g/L, sodium acetate 2-7g/L, dipotassium hydrogen phosphate 1-5g/L, Magnesium sulfate heptahydrate 0.25-0.65g/L, manganese sulfate monohydrate 0.1-0.3g/L, pH6.0-6.5.
8. the method that pomace according to claim 7 and cottonseed meal mixed fermentation produce albumen feedstuff, it is characterized in that: in step (4), fermentation to fermentation basal medium residual sugar content below 0.8% time, mending sugar with fermentation medium supplementing culture medium, after mending sugar, in culture fluid, residual sugar controls at 1-2%.
9. the method that pomace according to claim 8 and cottonseed meal mixed fermentation produce albumen feedstuff, it is characterised in that: described fermentation medium supplementing culture medium formula is glucose solution, and in described glucose solution, glucose content is the 2-4% of fermentation liquid total amount.
10. the method that pomace according to claim 1 and cottonseed meal mixed fermentation produce albumen feedstuff, it is characterised in that: stepIn, round selects check valve fermentation bag.
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| CN106509449A (en) * | 2016-12-30 | 2017-03-22 | 四川省旺达饲料有限公司 | Apple-pomace-fermented concentrated fiber product and preparation method thereof |
| CN106721028A (en) * | 2017-01-12 | 2017-05-31 | 江苏富海生物科技有限公司 | A kind of milk cow fermented feed and preparation method thereof |
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| CN101352198A (en) * | 2008-08-15 | 2009-01-28 | 天津生机集团股份有限公司 | Method for preparing Enterococcus faecalis fermented mixed feed for birds |
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Cited By (8)
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| CN106509449A (en) * | 2016-12-30 | 2017-03-22 | 四川省旺达饲料有限公司 | Apple-pomace-fermented concentrated fiber product and preparation method thereof |
| CN106721028A (en) * | 2017-01-12 | 2017-05-31 | 江苏富海生物科技有限公司 | A kind of milk cow fermented feed and preparation method thereof |
| CN106721028B (en) * | 2017-01-12 | 2020-05-22 | 江苏富海生物科技有限公司 | Fermented feed for dairy cows and preparation method thereof |
| CN107173544A (en) * | 2017-06-07 | 2017-09-19 | 江苏久久和牧农牧科技有限公司 | A kind of production method of miscellaneous dregs of rice fermented feed |
| CN111021104A (en) * | 2019-12-12 | 2020-04-17 | 苏州麻朵纺织科技有限公司 | Natural dyeing auxiliary for dyeing medium and light color series and dyeing method thereof |
| CN111436542A (en) * | 2020-04-02 | 2020-07-24 | 安徽天隆饲料有限公司 | Feed additive capable of replacing antibiotics to prevent diarrhea of chicks and improve growth and application of feed additive |
| CN112136618A (en) * | 2020-10-30 | 2020-12-29 | 广西禾美生态农业股份有限公司 | Chinese cabbage planting method |
| WO2023057314A1 (en) * | 2021-10-04 | 2023-04-13 | Omya International Ag | Composition comprising a surface-reacted calcium carbonate and a tannin |
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